The regulation of the cell cycle is governed and controlled by specific proteins, which are activated and deactivated mainly through phosphorylation/dephosphorylation processes in a precisely-timed manner. The key proteins that coordinate the initiation, progression, and completion of cell-cycle program are cyclin dependent kinases (CDKs). Cyclin-dependent kinases belong to the serine-threonine protein kinase family. They are heterodimeric complexes composed of a catalytic kinase subunit and a regulatory cyclin subunit. CDK activity is controlled by association with their corresponding regulatory subunits (cyclins) and CDK inhibitor proteins (Cip & Kip proteins, INK4s), by their phosphorylation state, and by ubiquitin-mediated proteolytic degradation (see D. G. Johnson, C. L. Walker, Annu. Rev. Pharmacol. Toxicol 39 (1999) 295-312; D. O. Morgan, Annu. Rev. Cell Dev. Biol. 13 (1997) 261-291; C. J. Sherr, Science 274 (1996) 1672-1677; T. Shimamura et al., Bioorg. Med. Chem. Lett. 16 (2006) 3751-3754).
There are four CDKs that are significantly involved in cellular proliferation: CDK1, which predominantly regulates the transition from G2 to M phase, and CDK2, CDK4, and CDK6, which regulate the transition from G1 to S phase (Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 2009; 9(3):153-166). In early to mid G1 phase, when the cell is responsive to mitogenic stimuli, activation of CDK4-cyclin D and CDK6-cyclin D induces phosphorylation of the retinoblastoma protein (pRb). Phosphorylation of pRb releases the transcription factor E2F, which enters the nucleus to activate transcription of other cyclins which promote further progression of the cell cycle (see J. A. Diehl, Cancer Biol. Ther. 1 (2002) 226-231; C. J. Sherr, Cell 73 (1993) 1059-1065). CDK4 and CDK6 are closely related proteins with basically indistinguishable biochemical properties (see M. Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630-641).
A number of CDK 4/6 inhibitors have been identified, including specific pyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas, benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, and oxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer Drug Targets 8 (2008) 53-75). For example, WO 03/062236 identifies a series of 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rb positive cancers that show selectivity for CDK4/6, including 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one (PD0332991), which is currently being tested by Pfizer in late stage clinical trials as an anti-neoplastic agent against estrogen-positive, HER2-negative breast cancer. Tate, et al. describe the antitumor activity of the CDK4/6 inhibitor abemaciclib (LY2835219) (“Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of the Antitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6 Inhibitor, in Mice Bearing Human Tumor Xenografts”, Clin Cancer Res (Jul. 15, 2014) 20; 3763). Rader, et al. describe the reduced proliferation in neuroblastoma-derived cell lines using the CDK4/6 inhibitor ribociclib (LEE011) (“Dual CDK4/CDK6 Inhibition Induces Cell Cycle Arrest and Senescence in Neuroblastoma”, Clin Cancer Res (Nov. 15, 2013) 19(22): 6173-82). VanderWel et al. describe an iodine-containing pyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4 inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387). WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase and serine/threonine kinase inhibitors. WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidine compounds as CDK inhibitors. WO 2011/101409 also filed by Novartis describes pyrrolopyrimidines with CDK 4/6 inhibitory activity. WO 2005/052147 filed by Novartis and WO 2006/074985 filed by Janssen Pharma disclose additional CDK4 inhibitors. WO 2012/061156 filed by Tavares and assigned to G1 Therapeutics describes CDK inhibitors. WO 2013/148748 filed by Francis Tavares and assigned to G1 Therapeutics describes Lactam Kinase Inhibitors. PCT Patent Application No. PCT/US2014/029073 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of hematopoietic stem and progenitor cells against ionizing radiation using CDK4/6 inhibitors. In one aspect, PCT/US2014/029073 describes the use of a CDK4/6 inhibitor to protect hematopoietic stem and progenitor cells in a subject with small cell lung cancer undergoing treatment with standard of care chemotherapeutics such as carboplatin, cisplatin, etoposide, topotecan, camptothecin, and irinotecan. PCT Patent Application No. PCT/US2014/028685 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of normal cells during chemotherapy using CDK4/6 inhibitors. PCT Patent Application No. PCT/US2014/029429 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for treating Rb-positive cancers using CDK4/6 inhibitors. PCT Patent Application No. PCT/US2014/029274 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for treating certain cancers with CDK4/6 inhibitors.
Selective CDK4/6 inhibitors are generally designed to target CDK4/6-replication dependent cancers. For example, Michaud et al., reported that the CDK4/6 inhibitor PD-0332991 was inactive against Rb-negative tumors. (Michaud et al., Pharmacologic inhibition of cyclin-dependent kinase 4 and 6 arrests the growth of glioblastoma multiform intracranial xenografts. Cancer Res. 70:3228-3238 (2010)).
Topoisomerase enzymes play a vital role in cellular proliferation and replication, altering the supercoiling of double-stranded DNA by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. DNA strand separation is obligatory to transcribe and replicate genomes by copying each base by RNA and DNA polymerases. (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433). Because of DNA's double-helical structure, replication generated catenated progenies that have to be unlinked by topoisomerases prior to cytokinesis.
Topoisomerases are classified as type I and type II. Type I enzymes cleave one DNA strand at a time and type II both strands to perform their catalytic functions. All topoisomerases cleave the DNA phosphodiester backbone by nucleophilic attack from a catalytic tyrosine residue which becomes linked to the phosphate end (P-Y) of the DNA break. Those reactions are highly reversible and leave the DNA sequence unchanged following topoisomerization (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).
A number of topoisomerase type I (Top1) inhibitors have been evaluated as anticancer therapeutics. Camptothecin was first identified from the Chinese tree Camptotheca acuminate (Wall et al., “The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminate,” J. Am. Chem. Soc. (1966) 88: 3888-3890.) A number of camptothecin derivatives, including for example topotecan, irinotecan, belotecan, gimatecan, lurtotecan, diflomotecan, S39625, and exatecan, have been further investigated as anticancer agents (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).
In addition to the camptothecin derivatives, several non-camptothecin topoisomerase inhibitors have also been investigated as anticancer agents, including the indolocarbazole edotecarin, indenoisoquinolines NSC 706744 (MJ-III-65), NSC 725776 (LMP-776), and NSC 724998 (LMP-400), dibenzonaphthyridiones such as topovale (ARC-111), and the aromathecin rosettacin (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).
In addition to the Top1 inhibitors, a number of anti-cancer agents have been investigated that target topoisomerase type II (Top2) enzymes, including etoposide, teniposide, and the DNA intercalators doxorubicin, daunorubicin, aclarubicin, amsacrine, dexrazoxane, TAS-103, the quinolone CP-115,963, the ellipticines including ellipticinium, azatoxins, genistein, VP-16, VM-26, mitoxantrone, amonafidem, and saintopin.
One potential side-effect of the use of topoisomerase inhibitors as anti-cancer agents includes the development of secondary malignancies. For example, the use of etoposide induces treatment-related acute myelocytic leukemia (t-AML) and treamtent related myelodysplastic syndromes (t-MDS), which often progress to t-AML (Pedersen-Bjergaard et al., “Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia,” Blood (2002) 99:1909-1912).
Previous studies have examined the potential cytotoxic activity of camptothecin derivatives in combination with CDK inhibitors against small cell lung cancer cell lines. Hamilton et al. found that the non-specific pan-CDK inhibitors olomoucine, roscovitine, and CDK4I had a synergistic cytotoxic effect on small cell lung cancer cell lines in combination with topotecan (“Synergistic Anticancer Activity of Topotecan—Cyclin-Dependent Kinase Inhibitor Combinations against Drug-Resistant Small Cell Lung Cancer (SCLC) Cell Lines”, Journal of Cancer Therapy (2013) 4: 47-53). In additional experiments, Hamilton et al. found that while the pan-CDK inhibitors olomoucine, roscovitine, and CDK4I had a synergistic cytotoxic effect on small cell lung cancer cell lines in combination with various camptothecin derivatives (including rubitecan, 9AC, topotecan, SN38, and CPT109), comparatively, the CDK 4/6 inhibitor PD0332991 had low chemosensitizing activity (“Synergism of Cyclin-Dependent Kinase Inhibitors with Camptothecin Derivatives in Small Cell Lung Cancer Cell Lines” Molecules (2014), 19(2): 2077-2088).
Accordingly, there is an ongoing need for improved compounds, methods, and regimes to treat patients with Rb-negative cancers and abnormal cellular proliferative disorders.